Blanton J R, Bidwell C A, Sanders D A, Sharkey C M, McFarland D C, Gerrard D E, Grant A L
Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA.
J Anim Sci. 2000 Apr;78(4):909-18. doi: 10.2527/2000.784909x.
Cell-mediated gene transfer is a potential tool for studying muscle growth, but efficient genetic manipulation and implantation strategies have not been developed for pigs. The objectives of the present study were to determine methods for transient and stable incorporation of reporter genes into porcine muscle cells and to investigate their use for cell-mediated gene transfer in pigs. Porcine myoblasts and fibroblasts were isolated from muscle of 2-wk-old male pigs. Myogenic cell lines were identified using muscle-specific monoclonal antibodies, myotube fusion assays, and the presence of muscle-specific markers (MyoD and desmin). Four commercial cationic liposomes (lipofectAMINE, lipofectin, cellFECTIN, and DMRIE-C) were tested at different DNA:lipid ratios for their ability to transfect myoblasts and fibroblasts transiently with a luciferase reporter plasmid. LipofectAMINE resulted in the greatest (P < .01) transient luciferase activity for both cell types. Electroporation of cells for transient transfection resulted in less luciferase activity than cationic transfection. Stable transfections were conducted using a green fluorescence protein (GFP) reporter plasmid containing the neomycin resistance gene. LipofectAMINE transfection resulted in stable GFP expression in 1:16,000 myoblasts and 1:33,000 fibroblasts. Stable electroporation resulted in efficiencies that were significantly lower than established with cationic liposomes. Porcine cells were transduced with GFP using vesicular stomatitis virus glycoprotein G pseudotyped retrovirus and resulted in efficiencies of 1:1.2 for myoblasts and 1:1.1 for fibroblasts. These results show that cationic liposomes are superior to electroporation for transfection, but retroviral transduction produced stable reporter gene expression in > 80% of porcine muscle cells. Transduced GFP-positive cells were separated from GFP-negative cells by fluorescence-activated cell sorting and implanted into 2-wk-old male pigs. On d 4, implanted muscles were removed and subjected to immunodetection of GFP protein. Fibroblast implantation resulted in limited GFP expression within muscle, whereas myoblast implantation resulted in GFP within muscle fibers. This suggests that cell-mediated gene transfer is possible in porcine muscle and may be useful as an approach for studying muscle growth in pigs.
细胞介导的基因转移是研究肌肉生长的一种潜在工具，但尚未为猪开发出高效的基因操作和植入策略。本研究的目的是确定将报告基因瞬时和稳定整合到猪肌肉细胞中的方法，并研究其在猪细胞介导的基因转移中的应用。从2周龄雄性猪的肌肉中分离出猪成肌细胞和平滑肌细胞。使用肌肉特异性单克隆抗体、肌管融合试验以及肌肉特异性标志物（MyoD和结蛋白）的存在来鉴定成肌细胞系。测试了四种商业阳离子脂质体（脂质体转染试剂、脂质体、细胞转染试剂和DMRIE-C）在不同DNA:脂质比例下用荧光素酶报告质粒瞬时转染成肌细胞和平滑肌细胞的能力。脂质体转染试剂对两种细胞类型均产生了最大的（P <.01）瞬时荧光素酶活性。细胞电穿孔用于瞬时转染时产生的荧光素酶活性低于阳离子转染。使用含有新霉素抗性基因的绿色荧光蛋白（GFP）报告质粒进行稳定转染。脂质体转染试剂转染导致在1:16,000的成肌细胞和1:33,000的平滑肌细胞中稳定表达GFP。稳定电穿孔产生的效率明显低于阳离子脂质体。使用水泡性口炎病毒糖蛋白G假型逆转录病毒用GFP转导猪细胞，成肌细胞的效率为1:1.2，平滑肌细胞的效率为1:1.1。这些结果表明，阳离子脂质体在转染方面优于电穿孔，但逆转录病毒转导在超过80%的猪肌肉细胞中产生了稳定的报告基因表达。通过荧光激活细胞分选将转导的GFP阳性细胞与GFP阴性细胞分离，并植入2周龄雄性猪体内。在第4天，取出植入的肌肉并进行GFP蛋白的免疫检测。平滑肌细胞植入导致肌肉内GFP表达有限，而成肌细胞植入导致肌肉纤维内出现GFP。这表明细胞介导的基因转移在猪肌肉中是可行的，并且可能作为研究猪肌肉生长的一种方法。
